Recent developments of recombinant DNA techniques and advanced peptide synthetic methods have permitted the commercial production of medically useful quantities of therapeutic polypeptides. The short half-life of many therapeutic polypeptides, however, has historically posed a challenge to the administration of these compounds. There are several important polypeptide-based drugs currently in use which would benefit from increased half-life. Examples are erythropoietin, insulin, interferon α-2b, interferon β, interferon γ, granulocyte colony stimulating factor, human growth hormone, granulocyte macrophage colony stimulating factor, relaxin, urokinase, streptokinase, tissue plasminogen activator, calcitonin, interleukin-2 and tumor necrosis factor with half-lives (significantly) less than a few hours. Insulin, for example, has a half-life of only about 12 minutes in man. Other examples of polypeptides that are being developed as potential therapeutic agents but suffer from short half-lives are adrenomedullin, glucagon like peptide (GLP-1) and kisspeptin (metastin). Extending the half-life of therapeutic polypeptides can improve current treatment by allowing dosing amounts and frequency of dosing to be reduced (Curr. Opin. Drug Disc. Dev. 2005, 8, 590-600).
Many proteins have already been subjected to studies aimed at extending the in vivo half-life employing e.g. adaptation by PEGylation (i.e. conjugation with a ˜1-30 kDa polyethylene glycol-moiety; Drug Discovery Today 2005, 10, 1451-1458). Currently available are for example PEGylated analogs of insulin with an extended half-life. Beside the reduced clearance rate, important aspects of the latter insulin derivatives are the reduced immunogenicity (e.g. U.S. Pat. No. 4,179,337) and increased solubility. Further, developments in PEGylation of insulin also led to physically and proteolytically more stable conjugates than native insulin (see for example WO 2004/091494, WO 2002/098232, US 2005/0152848).
PEGylated erythropoietin with a longer serum half-life is for example described in WO 2004/022577. It has further been found that by altered glycosylation of erythropoietin, the half-life increases. In addition, hyperglycosylated analogs of erythropoietin were reported to have higher in vivo activity (WO 2000/24893). Other examples of PEGylated (poly)peptides with prolonged duration of action are glucagon-like peptide-1 (GLP-1) (WO 2005/058954, WO 2004/093823; Bioconjugate Chem. 2005, 16, 377-382; Biomaterials, 2005, 26, 3597-3606), glucose-dependent insulinotropic polypeptide (GIP) (Bioorg. Med. Chem. Lett., 2005, 15, 4114-4117), calcitonin (Pharm. Dev. Technol. 1999, 4, 269-275) and octreotide (Pharm. Res. 2005, 22, 743-749).
Still, the use of PEG has limitations. PEG is obtained by chemical synthesis and, like all synthetic polymers, is polydisperse. This means that a batch of PEG consists of molecules having different numbers of monomers, resulting in a Gaussian distribution of the molecular weights. When a polypeptide is PEGylated, this leads to a collection of conjugates, which may have different biological properties, in particular in half-lives and immunogenicity. Reproducibility of the pharmacological activities of PEGylated polypeptides may therefore be a serious drawback of the technique. Also, it is known that PEGylation of proteins is often accompanied by loss of biological activity. Further, the use of PEG may cause problems relating to excretion from the body. At high molecular weights PEGs can accumulate in the liver, leading to macromolecular syndrome. Consequently, PEGylation of drugs should be performed with great care.
Similar results as with PEGylation were obtained by derivatization of polypeptides with polysaccharides, in particular with polysialic acid chains (e.g. WO 92/22331 and WO 2001/87922).
In JP 02/231077, heparin-superoxide dismutase (SOD) conjugates are described. Preferably, a number of heparin molecules are attached to SOD resulting in conjugates having a longer half-life than native SOD while retaining about 90% of the enzymatic activity.
Other conjugates of polypeptides with increased half-life are exemplified by conjugated derivatives of insulin (WO 2003/013573, WO 05/012346) or GLP-1 (Bioorg. Med. Chem. Lett. 2004, 14, 4395-4398) which bind to circulating serum albumin. The binding to serum albumin in those compounds is based in particular on hydrophobic interactions of the binding moiety within the conjugate with human serum albumin. The higher the hydrophobicity of that moiety, the stronger the binding affinity to human serum albumin. Although a wide range of binding moieties is suitable, a drawback of such conjugates is the low affinity and selectivity of the interaction of the conjugates with human serum albumin with as a result a poor predictability of the pharmacodynamic behavior. Alternatively, fusing the gene for human insulin directly to that for human serum albumin results in a long-acting form of insulin that is active in reducing blood glucose levels for a prolonged period after subcutaneous administration (Duttaroy et al. Diabetes 2005, 54, 251-258). However, in this case the bioavailability of the fused polypeptide, as well as the binding affinity for the target receptor, is reduced.
Further, in WO 2000/40253 conjugates of, for instance, a peptide and, specifically, glycosaminoglycan chain(s) are disclosed, which are considered as synthetic proteoglycans. In those conjugates the pharmacological activity of the conjugated glycosaminoglycan has a significant impact on the therapeutic activity of the conjugates.
Also, oligosaccharides are attached to pharmaceutically active compounds in order to increase the solubility thereof (WO 2004/03971).